Usefulness of circulating EPAC1 as biomarkers of therapeutic response to GLP-1 receptor agonists.

AIMS: The response to Glucagon-like peptide-1 receptor agonists (GLP-1RAs) is highly varia-ble among patients. Thus, the identification of predictive biomarkers of therapeutic response to GLP-1 RA could help us to optimize the use of this class of drugs. GLP-1RAs increase exchange proteins directly activated by cAMP (EPAC). The aim of the present study was to assess whether the increase of EPAC1 after GLP-1RAs treatment could be a biomarker of clinical response. METHODS: After showing that GLP-1 (10 ng/mL) significantly increased the expression of EPAC1 in human endo-thelial vascular cells (HUVEC), a pilot clinical study was planned. For this purpose 49 patients with type 2 diabetes who started treatment with liraglutide were included. EPAC1 concentration was determined by ELISA before and at one month of liraglutide treatment. RESULTS: We found that serum concentration of EPAC1 increased significantly after treatment with liraglutide. Only in those patients in whom EPAC1 increased (64%), a significant decrease in HbA1c, LDL-C, body mass index (BMI), and waist circumference was shown. CONCLUSIONS: This pilot study suggests that the increase of circulating EPAC1 after GLP-1RAs treatment could be a useful biomarker to predict clinical GLP1-RAs response.

Neuronal Dysfunction Is Linked to the Famine-Associated Risk of Proliferative Retinopathy in Patients With Type 2 Diabetes.

Persons with type 2 diabetes born in the regions of famine exposures have disproportionally elevated risk of vision-threatening proliferative diabetic retinopathy (PDR) in adulthood. However, the underlying mechanisms are not known. In the present study, we aimed to investigate the plausible molecular factors underlying progression to PDR. To study the association of genetic variants with PDR under the intrauterine famine exposure, we analyzed single nucleotide polymorphisms (SNPs) that were previously reported to be associated with type 2 diabetes, glucose, and pharmacogenetics. Analyses were performed in the population from northern Ukraine with a history of exposure to the Great Ukrainian Holodomor famine [the Diagnostic Optimization and Treatment of Diabetes and its Complications in the Chernihiv Region (DOLCE study), n = 3,583]. A validation of the top genetic findings was performed in the Hong Kong diabetes registry (HKDR, n = 730) with a history of famine as a consequence of the Japanese invasion during WWII. In DOLCE, the genetic risk for PDR was elevated for the variants in ADRA2A, PCSK9, and CYP2C19*2 loci, but reduced at PROX1 locus. The association of ADRA2A loci with the risk of advanced diabetic retinopathy in famine-exposed group was further replicated in HKDR. The exposure of embryonic retinal cells to starvation for glucose, mimicking the perinatal exposure to famine, resulted in sustained increased expression of Adra2a and Pcsk9, but decreased Prox1. The exposure to starvation exhibited a lasting inhibitory effects on neurite outgrowth, as determined by neurite length. In conclusion, a consistent genetic findings on the famine-linked risk of ADRA2A with PDR indicate that the nerves may likely to be responsible for communicating the effects of perinatal exposure to famine on the elevated risk of advanced stages of diabetic retinopathy in adults. These results suggest the possibility of utilizing neuroprotective drugs for the prevention and treatment of PDR.

Liraglutide Improves Forced Vital Capacity in Individuals With Type 2 Diabetes: Data From the Randomized Crossover LIRALUNG Study.

To evaluate the effect of liraglutide, a glucagon-like peptide 1 receptor agonist, on pulmonary function and serum levels of surfactant protein D (SP-D) in type 2 diabetes. A double-blind, randomized, crossover, placebo-controlled clinical trial comprising 76 patients with a baseline forced expiratory volume in 1 s <90% of that predicted. Liraglutide was administered for 7 weeks (2 weeks of titration plus 5 weeks at 1.8 mg daily). This short duration was intentional to minimize weight loss as a potential confounding factor. Serum level of SP-D was used as a biomarker of alveolar-capillary barrier integrity. Liraglutide exerted a positive impact on forced vital capacity (FVC) in comparison with placebo (ΔFVC 5.2% of predicted [from 0.8 to 9.6]; P = 0.009). No differences in the other pulmonary variables were observed. Participants under liraglutide treatment also experienced a decrease in serum SP-D (P = 0.038). The absolute change in FVC correlated with final serum SP-D in participants receiving liraglutide (r = -0.313, P = 0.036). Stepwise multivariate regression analysis showed that final serum SP-D independently predicted changes in FVC. In conclusion, liraglutide increased FVC in patients with type 2 diabetes. This effect was associated with a significant decrease of circulating SP-D, thus pointing to a beneficial effect in the alveolar-capillary function.

ERM Complex, A Therapeutic Target for Vascular Leakage Induced by Diabetes.

BACKGROUND: Ezrin, radixin, and moesin (the ERM complex) interact directly with membrane proteins regulating their attachment to actin filaments. ERM protein activation modifies cytoskeleton organization and alters the endothelial barrier function, thus favoring vascular leakage. However, little is known regarding the role of ERM proteins in diabetic retinopathy (DR). OBJECTIVE: This study aimed to examine whether overexpression of the ERM complex exists in db/db mice and its main regulating factors. METHODS: 9 male db/db mice and 9 male db/+ aged 14 weeks were analyzed. ERM proteins were assessed by western blot and by immunohistochemistry. Vascular leakage was determined by the Evans blue method. To assess ERM regulation, HRECs were cultured in a medium containing 5.5 mM D-glucose (mimicking physiological conditions) and 25 mM D-glucose (mimicking hyperglycemia that occurs in diabetic patients). Moreover, treatment with TNF-α, IL-1ß, or VEGF was added to a high glucose condition. The expression of ERM proteins was quantified by RT-PCR. Cell permeability was evaluated by measuring movements of FITC-dextran. RESULTS: A significant increase of ERM in diabetic mice in comparison with non-diabetic mice was observed. A high glucose condition alone did not have any effect on ERM expression. However, TNF-α and IL-1ß induced a significant increase in ERM proteins. CONCLUSION: The increase of ERM proteins induced by diabetes could be one of the mechanisms involved in vascular leakage and could be considered as a therapeutic target. Moreover, the upregulation of the ERM complex by diabetes is induced by inflammatory mediators rather than by high glucose itself.

δ Opioid Receptor Agonism Preserves the Retinal Pigmented Epithelial Cell Tight Junctions and Ameliorates the Retinopathy in Experimental Diabetes.

Purpose: Outer blood retinal barrier breakdown is a neglected feature of diabetic retinopathy (DR). We demonstrated that the agonism of the δ opioid receptor (DOR) by epicatechin preserves the tight junction proteins in ARPE-19 cells under diabetic conditions. Presently, we aimed to evaluate the possible role of the DOR on the maintenance of tight junction of RPE layer and on the early markers of experimental DR. Methods: DR markers and external retinal tight junction proteins were evaluated in CL57B diabetic mice submitted to intravitreous injection of short hairpin RNA (shRNA)-DOR (108 transducing units [TU]/mL) treated or not with DOR agonist (0.05 g/animal/d of epicatechin in drinking water) for 16 weeks. The presence of DOR in human retina from postmortem eyes from diabetic and nondiabetic donors were also performed. Results: DOR is present in RPE layer and in neuro retina. The treatment with DOR agonist prevented the upregulation of the early markers of retinopathy (glial fibrillary acidic protein, VEGF) and the downregulation of pigment epithelium-derived factor, occludin, claudin-1, and zonula occludens-1 tight junction expressions. The silencing of DOR in retina of diabetic mice partially abolished the protective effects of epicatechin. In human retina specimens, DOR is present throughout the retina, similarly in nondiabetic and diabetic donors. Conclusions: This set of experiments strongly indicates that the DOR agonism preserves RPE tight junctions and reduces the early markers of retinopathy in model of diabetes. These novel findings designate DOR as a potential therapeutic tool to treat DR with preservation of the RPE tight junction proteins.

SOCS1-Derived Peptide Administered by Eye Drops Prevents Retinal Neuroinflammation and Vascular Leakage in Experimental Diabetes.

Current treatments for diabetic retinopathy (DR) target late stages when vision has already been significantly affected. Accumulating evidence suggests that neuroinflammation plays a major role in the pathogenesis of DR, resulting in the disruption of the blood-retinal barrier. Suppressors of cytokine signaling (SOCS) are cytokine-inducible proteins that function as a negative feedback loop regulating cytokine responses. On this basis, the aim of the present study was to evaluate the effect of a SOCS1-derived peptide administered by eye drops (2 weeks) on retinal neuroinflammation and early microvascular abnormalities in a db/db mouse model. In brief, we found that SOCS1-derived peptide significantly reduced glial activation and neural apoptosis induced by diabetes, as well as retinal levels of proinflammatory cytokines. Moreover, a significant improvement of electroretinogram parameters was observed, thus revealing a clear impact of the histological findings on global retinal function. Finally, SOCS1-derived peptide prevented the disruption of the blood-retinal barrier. Overall, our results suggest that topical administration of SOCS1-derived peptide is effective in preventing retinal neuroinflammation and early microvascular impairment. These findings could open up a new strategy for the treatment of early stages of DR.

Topical Administration of Bosentan Prevents Retinal Neurodegeneration in Experimental Diabetes.

Experimental evidence suggests that endothelin 1 (ET-1) is involved in the development of retinal microvascular abnormalities induced by diabetes. The effects of ET-1 are mediated by endothelin A- and B-receptors (ETA and ETB). Endothelin B-receptors activation mediates retinal neurodegeneration but there are no data regarding the effectiveness of ETB receptor blockage in arresting retinal neurodegeneration induced by diabetes. The main aim of the present study was to assess the usefulness of topical administration of bosentan (a dual endothelin receptor antagonist) in preventing retinal neurodegeneration in diabetic (db/db) mice. For this purpose, db/db mice aged 10 weeks were treated with one drop of bosentan (5 mg/mL, n = 6) or vehicle (n = 6) administered twice daily for 14 days. Six non-diabetic (db/+) mice matched by age were included as the control group. Glial activation was evaluated by immunofluorescence using specific antibodies against glial fibrillary acidic protein (GFAP). Apoptosis was assessed by TUNEL method. A pharmacokinetic study was performed in rabbits. We found that topical administration of bosentan resulted in a significant decrease of reactive gliosis and apoptosis. The results of the pharmacokinetic study suggested that bosentan reached the retina through the trans-scleral route. We conclude that topical administration of bosentan was effective in preventing neurodegeneration in the diabetic retina and, therefore, could be a good candidate to be tested in clinical trials.

Proteomic Analysis of Early Diabetic Retinopathy Reveals Mediators of Neurodegenerative Brain Diseases.

Purpose: Current evidence suggests that retinal neurodegeneration is an early event in the pathogenesis of diabetic retinopathy. Our main goal was to examine whether, in the diabetic human retina, common proteins and pathways are shared with brain neurodegenerative diseases. Methods: A proteomic analysis was performed on three groups of postmortem retinas matched by age: nondiabetic control retinas (n = 5), diabetic retinas without glial activation (n = 5), and diabetic retinas with glial activation (n = 5). Retinal lysates from each group were pooled and run on an SDS-PAGE gel. Bands were analyzed sequentially by liquid chromatography-mass spectrometry (LC/MS) using an Orbitrap Mass Spectrometer. Results: A total of 2190 proteins were identified across all groups. To evaluate the association of the identified proteins with neurological signaling, significant signaling pathways belonging to the category "Neurotransmitters and Other Nervous System Signaling" were selected for analysis. Pathway analysis revealed that "Neuroprotective Role of THOP1 in Alzheimer's Disease" and "Unfolded Protein Response" pathways were uniquely enriched in control retinas. By contrast, "Dopamine Degradation" and "Parkinson's Signaling" were enriched only in diabetic retinas with glial activation. The "Neuregulin Signaling," "Synaptic Long Term Potentiation," and "Amyloid Processing" pathways were enriched in diabetic retinas with no glial activation. Conclusions: Diabetes-induced retinal neurodegeneration and brain neurodegenerative diseases, such as Alzheimer's and Parkinson's diseases, share common pathogenic pathways. These findings suggest that the study of neurodegeneration in the diabetic retina could be useful to further understand the neurodegenerative processes that occur in the brain of persons with diabetes.

Silymarin prevents diabetes-induced hyperpermeability in human retinal endothelial cells / La silimarina previene la hiperpermeabilidad inducida por la diabetes en células endoteliales de retina humana

Introduction: Vascular endothelial growth factor (VEGF) plays an essential role in development of diabetic macular edema (DME). While there is evidence suggesting that silymarin, a flavonoid extracted from Silybum marianum, could be useful for prevention and treatment of diabetic nephropathy, no studies have been conducted in diabetic retinopathy (DR). The aim of this study was to assess the effect of silymarin on disruption of inner blood retinal barrier (BRB), the primary cause of DME. Materials and methods: Human retinal endothelial cells (HRECs) were cultured under standard (5.5mM D-glucose) and diabetogenic conditions (25mM D-glucose and 25mM D-glucose + recombinant vascular endothelial growth factor [rVEGF, 25mg/mL]). To assess cell viability, three concentrations of silymarin were tested (2, 4 and 10μg/mL). The effect of silymarin on HREC disruption was determined using a dextran (70kD) permeability asssay. Results: No differences were found in the viability of HRECs treated with 2 or 4μg/mL of silymarin as compared to untreated cells, but viability significantly decreased after using 10 μg/mL. The concentration of 4 μg/mL was therefore selected. Silymarin (4μg/mL) caused a significant decrease in VEGF-induced permeability in both media with 5.5nM (422±58 vs. 600±72 ng/mL/cm2; p<0.03) and 25nM of D-glucose (354 ± 28 vs. 567 ± 102 ng/mL/cm2; p<0.04). Discussion: Our results show that silymarin is effective for preventing hyperpermeability induced by diabetic conditions in HRECs. Further studies are needed to assess whether silymarin could be useful to treat DME (AU)

Introducción: El Vascular endothelial growth factor (VEGF) juega un papel esencial en el desarrollo del edema macular diabético (EMD). Existe evidencia que indica que el uso de la silimarina, extracto flavonoide del Silybum marianum, podría ser útil en la prevención y el tratamiento de la nefropatía diabética pero no se dispone de datos en retinopatía diabética (RD). El objetivo del estudio es evaluar el efecto de la silimarina sobre la disrupción de la barrera hematorretininana, que es la causa primaria del EMD. Material y métodos: Células endoteliales de retina humana (HRECs) se cultivaron en condiciones estándar (5.5mM de D-glucosa) y en condiciones suprafisiológicas de glucosa (25mM de D-glucosa y 25mM de D-glucosa + VEGF 25mg/dl). Para evaluar la viabilidad de las células se probaron 3 concentraciones de silimarina (2, 4 y 10μg/ml). El efecto de la silimarina sobre la disrupción de las HRECs se determinó mediante análisis de permeabilidad a dextrano (70kD). Resultados: No se observaron diferencias en la viabilidad de las HRECs tratadas con 2 o 4μg/ml de silimarina en comparación con las células no tratadas, pero se observó una reducción de la viabilidad con la concentración de 10μg/ml. Por consiguiente, se seleccionó la concentración de 4μg/ml de silimarina. La silimarina (4μg/ml) produjo un descenso significativo de la permeabilidad inducida por VEGF tanto en medio con 5.5mM de D-glucosa (422 ±58 vs. 600 ±72 ng/ml/cm2; p<0.03) como en medio con 25mM de D-glucosa (354±28 vs. 567±102 ng/ml/cm2; p<0.04). Discusión: Nuestros resultados demuestran que la silimarina es efectiva para prevenir la hiperpermeabilidad inducida por condiciones suprafisiológicas de glucosa en HRECs. Son necesarios más estudios para evaluar si la silimarina podría ser útil para el tratamiento del EMD (AU)

Silymarin prevents diabetes-induced hyperpermeability in human retinal endothelial cells.

INTRODUCTION: Vascular endothelial growth factor (VEGF) plays an essential role in development of diabetic macular edema (DME). While there is evidence suggesting that silymarin, a flavonoid extracted from Silybum marianum, could be useful for prevention and treatment of diabetic nephropathy, no studies have been conducted in diabetic retinopathy (DR). The aim of this study was to assess the effect of silymarin on disruption of inner blood retinal barrier (BRB), the primary cause of DME. MATERIALS AND METHODS: Human retinal endothelial cells (HRECs) were cultured under standard (5.5mM D-glucose) and diabetogenic conditions (25mM D-glucose and 25mM D-glucose + recombinant vascular endothelial growth factor [rVEGF, 25mg/mL]). To assess cell viability, three concentrations of silymarin were tested (2, 4 and 10µg/mL). The effect of silymarin on HREC disruption was determined using a dextran (70kD) permeability asssay. RESULTS: No differences were found in the viability of HRECs treated with 2 or 4µg/mL of silymarin as compared to untreated cells, but viability significantly decreased after using 10µg/mL. The concentration of 4 µg/mL was therefore selected. Silymarin (4µg/mL) caused a significant decrease in VEGF-induced permeability in both media with 5.5nM (422±58 vs. 600±72 ng/mL/cm2; p<0.03) and 25nM of D-glucose (354 ± 28 vs. 567 ± 102 ng/mL/cm2; p<0.04). DISCUSSION: Our results show that silymarin is effective for preventing hyperpermeability induced by diabetic conditions in HRECs. Further studies are needed to assess whether silymarin could be useful to treat DME.

Association of HMGB1 with oxidative stress markers and regulators in PDR.

Purpose: We investigated the link among the proinflammatory cytokine high-mobility group box 1 (HMGB1) and 8-hydroxy-2'-deoxyguanosine (8-OHdG) as a marker of oxidative DNA damage, the endothelial adhesion molecule and oxidase enzyme vascular adhesion protein-1 (VAP-1), and the inducible cytoprotective molecule heme oxygenase-1 (HO-1) in proliferative diabetic retinopathy (PDR). We correlated the levels of these molecules with clinical disease activity and studied the proinflammatory activities of HMGB1 on rat retinas and human retinal microvascular endothelial cells (HRMECs). Methods: Vitreous samples from 47 PDR and 19 non-diabetic patients, epiretinal membranes from 11 patients with PDR, human retinas (16 from diabetic patients and 16 from non-diabetic subjects), rat retinas, and HRMECs were studied by enzyme-linked immunosorbent assay, immunohistochemistry, western blot immunofluorescence, and RT-PCR analyses. In addition, we assessed the adherence of leukocytes to HMGB1-stimulated HRMECs. Results: HMGB1, 8-OHdG, and soluble VAP-1 (sVAP-1) levels were significantly higher in vitreous samples from PDR patients than in those from non-diabetics (p = 0.001, <0.0001, <0.0001, respectively). The HMGB1, 8-OHdG, sVAP-1, and HO-1 levels in PDR with active neovascularization were significantly higher than those in inactive PDR (p = 0.025, <0.0001, <0.0001, 0.012, respectively). Significant positive correlations were observed between the levels of HMGB1 and the levels of 8-OHdG (r = 0.422; p = 0.001) and sVAP-1 (r = 0.354; p = 0.004) and between the levels of 8-OHdG and the levels of sVAP-1 (r = 0.598; p<0.0001). In epiretinal membranes, VAP-1 and 8-OHdG were expressed in vascular endothelial cells and stromal cells. Significant increases in the VAP-1 mRNA and protein levels were detected in the RPE, but not in the neuroretina of diabetic patients. Treatment of HRMEC with HMGB1, diabetes induction, and an intravitreal injection of HMGB1 in normal rats induced a significant upregulation of the adhesion molecule intercellular adhesion molecule-1 (ICAM-1) in HRMECs and retinas. On the other hand, the expressions of vascular cell adhesion molecule-1 and VAP-1 were not affected. Oral administration of the HMGB1 inhibitor glycyrrhizin in rats attenuated the diabetes-induced upregulation of the retinal ICAM-1 expression. Treatment of HRMECs with HMGB1 increased leukocyte adhesion and induced the upregulation of 8-OHdG and HO-1 and the membranous translocation of VAP-1. Conclusions: Our results suggest a potential link among the proinflammatory cytokine HMGB1, VAP-1, oxidative stress, and HO-1 in the pathogenesis of PDR.

A compartmentalized microfluidic chip with crisscross microgrooves and electrophysiological electrodes for modeling the blood-retinal barrier.

The interconnection of different tissue-tissue interfaces may extend organ-on-chips to a new generation of sophisticated models capable of recapitulating more complex organ-level functions. Single interfaces are largely recreated in organ-on-chips by culturing the cells on opposite sides of a porous membrane that splits a chamber in two or by connecting the cells of two adjacent compartments through microchannels. However, it is difficult to interconnect more than one interface using these approaches. To address this challenge, we present a novel microfluidic device where cells are arranged in parallel compartments and are highly interconnected through a grid of microgrooves, which facilitates paracrine signaling and heterotypic cell-cell contact between multiple tissues. In addition, the device includes electrodes on the substrate for the measurement of transepithelial electrical resistance (TEER). Unlike conventional methods for measuring the TEER where electrodes are on each side of the cell barrier, a method with only electrodes on the substrate has been validated. As a proof-of-concept, we have used the device to mimic the structure of the blood-retinal barrier by co-culturing primary human retinal endothelial cells (HREC), a human neuroblastoma cell line (SH-SY5Y), and a human retinal pigment epithelial cell line (ARPE-19). Cell barrier formations were assessed by a permeability assay, TEER measurements, and ZO-1 expression. These results validate the proposed microfluidic device with microgrooves as a promising in vitro tool for the compartmentalization and monitoring of barrier tissues.

Serum Surfactant Protein D as a Biomarker for Measuring Lung Involvement in Obese Patients With Type 2 Diabetes.

Context: Lung impairment is a new target for late diabetic complications. Biomarkers that could help identify patients requiring functional respiratory tests have not been reported. Objective: Our aim was to examine whether serum surfactant protein D (SP-D) and A (SP-A) could be useful biomarkers of lung damage in obese patients with type 2 diabetes (T2D) without known lung disease. Design and Setting: A case-control study conducted in an ambulatory obesity unit. Patients: Forty-nine obese patients with T2D and 98 subjects without diabetes matched by age, sex, body mass index, and waist circumference were included. Interventions: Serum SP-D and SP-A levels were measured using enzyme-linked immunosorbent assay. Forced spirometry and static pulmonary volume were assessed. Results: Patients with T2D exhibited higher serum SP-D concentrations than control subjects (P = 0.006). No differences in serum SP-A concentrations were observed. There was an inverse association between forced expiratory volume in 1 second (FEV1) and serum SP-D (r = -0.265; P = 0.029), as well as a significant positive relationship between SP-D concentration and residual volume (r = 0.293; P = 0.043). From receiver operating characteristic analysis, the best SP-D cutoff to identify a FEV1 <80% of predicted was 132.3 ng/mL (area under the curve, 0.725; sensitivity, 77.7%; specificity, 69.4%). Stepwise multivariate regression analysis showed that serum SP-D concentration ≥132.3 ng/mL was independently associated with a FEV1 <80% of predicted (R2 = 0.406). Only the existence of T2D contributed independently to serum SD-P variance among all subjects (R2 = 0.138). Conclusions: Serum SP-D concentration can be a useful biomarker for detecting lung impairment in obese patients with T2D.

Calcium dobesilate prevents the oxidative stress and inflammation induced by diabetes in the retina of db/db mice.

AIM: Calcium dobesilate (CaD) is beneficial in early stages of diabetic retinopathy (DR), but its mechanisms of action remains to be elucidated. The aim was to investigate the effect of CaD on proinflammatory cytokines and oxidative stress. METHODS: db/db mice were randomly assigned to daily oral treatment with CaD (200mg/kg/day) or vehicle for 15days. Biomarkers of oxidative stress (dihydroethidium, malondialdehyde), NF-κB, and proinflammatory cytokines (IL-1ß, IL-6, IL-8, TNF-α, MCP-1) were examined in the retina by immunohistochemical analysis. Cultures of human retinal endothelial cells (HRECs) were used for complementary experiments. RESULTS: CaD significantly reduced the biomarkers of oxidative stress in the retina of db/db mice. In addition, CaD prevented the increase of NF-κB, IL-6, IL-8, TNF-α and MCP-1 induced by diabetes. CaD inhibited the activation of NF-kß induced by IL-1ß by preventing IKKB-α phosphorylation in HRECs and reduced the upregulation of IL-6 and IL-18 induced by TNF-α in a dose-dependent manner. CONCLUSION: Our results suggest that antioxidant and antiinflammatory effects are crucial in accounting for the effectiveness of CaD for treating DR.

Topical administration of DPP-IV inhibitors prevents retinal neurodegeneration in experimental diabetes.

AIMS/HYPOTHESIS: The main aims of the present study were: (1) to assess the expression and content of dipeptidyl peptidase IV (DPP-IV) in human and db/db mouse retinas, and in human vitreous fluid; and (2) to determine whether the topical administration of the DPP-IV inhibitors (DPP-IVi) would prevent retinal neurodegeneration and vascular leakage in db/db mice by reducing endogenous glucagon-like peptide 1 (GLP-1) degradation. METHODS: To assess the expression and content of DPP-IV, human samples of vitreous fluid and retinas were obtained from participants with type 2 diabetes (n = 8) and age-matched non-diabetic individuals (n = 8), as well as from db/db (n = 72) and db/+ (n = 28) mice. The interventional study, which included 72 db/db mice, consisted of the topical administration (eye drops) of saxagliptin, sitagliptin or vehicle for 14 days. DPP-IV mRNA levels were assessed by RT-PCR, and protein content was measured by ELISA or western blotting. GLP-1 was assessed by immunofluorescence, and its downstream effector exchange protein activated by cAMP-1 (EPAC-1) was used as a measure of GLP-1 receptor activation. Retinal analyses were performed in vivo by electroretinography and ex vivo by RT-PCR (Epac-1, Iba-1 [also known as Aif1]), western blotting (EPAC-1, glial fibrillar acidic protein [GFAP], glutamate-aspartate transporter [GLAST]) and immunofluorescence measurements (GLP-1, GFAP, ionised calcium binding adaptor molecule 1 [IBA-1], TUNEL, GLAST, albumin and collagen IV). Glutamate was quantified by HPLC. In addition, vascular leakage was examined by the Evans Blue method. RESULTS: DPP-IV was present in human vitreous fluid but in a range 100-fold less than in plasma. Both mRNA levels and protein content were much lower in the retina than in the liver or bowel, but were significantly higher in retinal pigment epithelium (RPE) from diabetic donors in comparison to non-diabetic donors (p < 0.05). Topical treatment with DPP-IVi prevented glial activation, apoptosis and vascular leakage induced by diabetes in db/db mice (p < 0.05). Moreover, it also significantly prevented diabetes-induced functional abnormalities in the electroretinogram. A significant increase of both GLP-1 and EPAC-1 was found after treatment with DPP-IVi (p < 0.05). Furthermore, GLAST downregulation induced by diabetes was prevented, resulting in a significant reduction of extracellular glutamate concentrations. All these effects were observed without any changes in blood glucose levels. CONCLUSIONS/INTERPRETATION: The topical administration of DPP-IVi is effective in preventing neurodegeneration and vascular leakage in the diabetic retina. These effects can be attributed to an enhancement of GLP-1, but other mechanisms unrelated to the prevention of GLP-1 degradation cannot be ruled out.

Calcium Dobesilate Prevents Neurodegeneration and Vascular Leakage in Experimental Diabetes.

PURPOSE: The mechanisms involved in the reported beneficial effects of Calcium dobesilate monohydrate (CaD) for the treatment of diabetic retinopathy (DR) remain to be elucidated. The main aim of the present study is to examine whether CaD prevents early events in the pathogenesis of DR such as neurodegeneration and vascular leakage. In addition, putative mediators of both neurodegeneration (glutamate/GLAST, ET-1/ETB receptor) and early microvascular impairment (ET-1/ETA receptor, oxidative stress, VEGF, and the PKC-delta-p38 MAPK pathway) have been examined. METHODS: Diabetic (db/db) mice were randomly assigned to daily oral treatment with CaD (200 mg/Kg/day) (n = 12) or vehicle (n = 12) for 14 days. In addition, 12 non-diabetic (db/+) mice matched by age were used as the control group. Functional abnormalities were assessed by electroretinography. Neurodegeneration and microvascular abnormalities were evaluated by immunohistochemistry and Western blot. Glutamate was determined by HPLC. RESULTS: CaD significantly decreased glial activation and apoptosis and produced a significant improvement in the electroretinogram parameters. Mechanistically, CaD prevented the diabetes-induced up-regulation of ET-1 and its cognate receptors (ETA-R and ETB-R), which are involved in microvascular impairment and neurodegeneration, respectively. In addition, treatment with CaD downregulated GLAST, the main glutamate transporter, and accordingly prevented the increase in glutamate. Finally, CaD prevented oxidative stress, and the upregulation of VEGF and PKC delta-p38 MAPK pathway induced by diabetes, thus resulting in a significant reduction in vascular leakage. CONCLUSIONS: Our findings demonstrate for the first time that CaD exerts neuroprotection in an experimental model of DR. In addition, we provide first evidence that CaD prevents the overexpression of ET-1 and its receptors in the diabetic retina. These beneficial effects on the neurovascular unit could pave the way for clinical trials addressed to confirm the effectiveness of CaD in very early stages of DR.

geoRge: A Computational Tool To Detect the Presence of Stable Isotope Labeling in LC/MS-Based Untargeted Metabolomics.

Studying the flow of chemical moieties through the complex set of metabolic reactions that happen in the cell is essential to understanding the alterations in homeostasis that occur in disease. Recently, LC/MS-based untargeted metabolomics and isotopically labeled metabolites have been used to facilitate the unbiased mapping of labeled moieties through metabolic pathways. However, due to the complexity of the resulting experimental data sets few computational tools are available for data analysis. Here we introduce geoRge, a novel computational approach capable of analyzing untargeted LC/MS data from stable isotope-labeling experiments. geoRge is written in the open language R and runs on the output structure of the XCMS package, which is in widespread use. As opposed to the few existing tools, which use labeled samples to track stable isotopes by iterating over all MS signals using the theoretical mass difference between the light and heavy isotopes, geoRge uses unlabeled and labeled biologically equivalent samples to compare isotopic distributions in the mass spectra. Isotopically enriched compounds change their isotopic distribution as compared to unlabeled compounds. This is directly reflected in a number of new m/z peaks and higher intensity peaks in the mass spectra of labeled samples relative to the unlabeled equivalents. The automated untargeted isotope annotation and relative quantification capabilities of geoRge are demonstrated by the analysis of LC/MS data from a human retinal pigment epithelium cell line (ARPE-19) grown on normal and high glucose concentrations mimicking diabetic retinopathy conditions in vitro. In addition, we compared the results of geoRge with the outcome of X(13)CMS, since both approaches rely entirely on XCMS parameters for feature selection, namely m/z and retention time values. To ensure data traceability and reproducibility, and enabling for comparison with other existing and future approaches, raw LC/MS files have been deposited in MetaboLights (MTBLS213) and geoRge is available as an R script at https://github.com/jcapelladesto/geoRge.

Topical Administration of GLP-1 Receptor Agonists Prevents Retinal Neurodegeneration in Experimental Diabetes.

Retinal neurodegeneration is an early event in the pathogenesis of diabetic retinopathy (DR). Since glucagon-like peptide 1 (GLP-1) exerts neuroprotective effects in the central nervous system and the retina is ontogenically a brain-derived tissue, the aims of the current study were as follows: 1) to examine the expression and content of GLP-1 receptor (GLP-1R) in human and db/db mice retinas; 2) to determine the retinal neuroprotective effects of systemic and topical administration (eye drops) of GLP-1R agonists in db/db mice; and 3) to examine the underlying neuroprotective mechanisms. We have found abundant expression of GLP-1R in the human retina and retinas from db/db mice. Moreover, we have demonstrated that systemic administration of a GLP-1R agonist (liraglutide) prevents retinal neurodegeneration (glial activation, neural apoptosis, and electroretinographical abnormalities). This effect can be attributed to a significant reduction of extracellular glutamate and an increase of prosurvival signaling pathways. We have found a similar neuroprotective effect using topical administration of native GLP-1 and several GLP-1R agonists (liraglutide, lixisenatide, and exenatide). Notably, this neuroprotective action was observed without any reduction in blood glucose levels. These results suggest that GLP-1R activation itself prevents retinal neurodegeneration. Our results should open up a new approach in the treatment of the early stages of DR.

Fenofibrate prevents the disruption of the outer blood retinal barrier through downregulation of NF-κB activity.

AIMS: There is clinical evidence that fenofibrate, a PPARα agonist, arrests the progression of diabetic macular edema (DME). However, the underlying mechanisms of this beneficial effect remain to be elucidated. We previously reported that fenofibric acid (FA), the active metabolite of fenofibrate, prevents the disorganization of tight junction proteins and the hyperpermeability provoked by the diabetic milieu in the retinal pigment epithelium (RPE). The aim of the present study was to evaluate whether this effect is mediated by inhibiting the proinflammatory transcription factor NF-κB, as well as the expression of several proinflammatory cytokines involved in the pathogenesis of DME. METHODS: Human RPE cells were cultured under standard conditions and under conditions leading to the disruption of the monolayer [IL-1ß (10 ng/ml)]. The effect of FA, QNZ (a NF-κB inhibitor), WY14643 (a PPARα agonist), and MK-866 (a PPARα antagonist) in the disruption of the monolayer was determined by dextran permeability and immunohistochemistry analyses. The effect of FA on NF-κB activity was assessed by EMSA and by NF-κB/p65 nuclear translocation analyses. The expression of cytokines (IL-6, IL-8, MCP-1) was measured by RT-PCR. RESULTS: FA prevented RPE monolayer disruption, and the consequent hyperpermeability induced by IL-1ß, through inhibition of NF-κB activity. This effect was due to PPARα activation and was associated with a significant downregulation of the expression of proinflammatory cytokines. CONCLUSIONS: Our findings suggest that the anti-inflammatory effects of FA through inhibition of NF-κB activity play a key role in the beneficial effect of fenofibrate for treating DME.